Project summary. Biological regulation depends on protein allostery, in which a perturbation at one site in a protein causes a functional change at a distal site. Because characterization of allostery challenges the limits of our technical abilities ? requiring simultaneous observation of changes in the structure, dynamics, function and energetics of a protein ensemble ? few allosteric mechanisms are understood in atomistic detail. This knowledge gap limits our fundamental understanding of how allostery is conserved, evolved, or affected in disease, as well as our abilities to design new allosteric proteins. Bridging the gap requires methods to map, validate and tune specific interactions that drive proteins to shift their equilibria to occupy different functional states. I am an NIH IRACDA and UCSF Chancellor's Postdoctoral fellow at UCSF. My mentor is Dr. Tanja Kortemme, an expert in computational biophysics. In the last three years I engineered and characterized an artificial protein biosensor using computational protein design methods and biophysical techniques. This work is the first example of the de novo design of a small molecule binding site in a protein-protein interface to build a functional, modular sensor/actuator system. In this proposal, I aim to establish a new platform for characterizing and engineering signal transduction in allosteric proteins. With the guidance of my co-mentor Dr. Susan Marqusee, a pioneer in hydrogen exchange methods, I propose in Aim 1 to map allosteric and energetic coupling among protein residues, ligands and solvent in the lac repressor (LacI) using hydrogen-deuterium exchange with mass spectrometry (HX/MS) and isothermal titration calorimetry. With the mentorship of Dr. Kortemme, I will computationally test this mechanism by reengineering LacI to switch its allosteric response to different ligands, establishing a strategy to control conformational equilibria in proteins. In Aim 2, I propose to extend this approach to proteins in the LacI/GalR family of sugar-responsive transcription factors to determine and validate to what extent allostery is conserved among structurally and functionally related proteins. In Aim 3, I will apply our platform to characterize the evolution and regulation of allostery in an ancient, highly conserved protein, and learn how cancer-associated mutations impact its mechanism. With the support of my mentors, collaborators, and the greater research environment at UCSF and UC Berkeley, I will train in HX/MS, computational methods development, quantitative analysis of large datasets, library construction, and high- throughput screening. These skills will help me bridge my background in biochemical engineering with my fascination with protein regulation to build a successful independent research program investigating the fundamentals principles that govern allostery, and engineering new therapeutic proteins.